DESCRIPTION: The branched-chain sugars are an important class of carbohydrates found widely in nature. They are formally derived from common sugars by replacement of either a hydrogen or a hydroxyl group on a secondary carbon atom with an alkyl side chain. Such a substitution generally causes a critical alteration of the biological function of the resulting sugar, and also induces a fundamental change in its metabolism. Particularly notable are the branched-chain sugars found in many antibiotics in which these unusual sugars play an indispensable role in conferring optimal activity on these natural products. Although the biological importance of branched-chain sugars is well recognized, little is known about the biosynthesis pathways leading to the formation of two branched-chain sugars, yersiniose A and dihydrostreptose. Yersiniose A is an immunodominant sugar found in the lipopolysaccharide (LPS) of Yersinia pseudotuberculosis VI, and dihydrostreptose is a structural component of streptomycin antibiotics isolated from Streptomyces griseus. Emphasis will be placed on the mechanistic studies of enzymes catalyzing the branched-chain construction steps in both cases. The proposed experiments include: 1) to clone the genes encoding the target enzymes and express these genes in E. coli to give catalytically active proteins; 2) to develop appropriate methods to assay the activity of the target enzymes; 3) to purify the target enzymes and characterize their physical and biochemical properties; 4) to prepare alternative substrates as mechanistic probe to study these enzymatic reactions; 5) to elucidate the detailed reaction mechanisms of these enzymes. An understanding of the molecular basis of the biosynthetic formation of these branched-chain sugars will not only aid in delineating how chemical transformations are effected by enzymes catalyzing these conversions, but will also provide invaluable knowledge for designing approaches to control and/or mimic their production and biological activities. Since the significance of sugar residues in determining the biological activity of the antibiotics has been well established, and some of the glycosyl transferases involved in the biosynthesis of antibiotics have been shown to have somewhat relaxed substrate specificity, it is expected that the new insights gained from these studies will also lay groundwork for gene transfer experiments to produce novel or hybrid antibiotics.